Abstract

   Epidemiological studies show that some diabetic patients develop
   end‐stage renal dysfunction without significant proteinuria or
   glomerulopathy, underscoring the role of renal tubular epithelial cell
   (TEC) impairment in diabetic kidney disease (DKD). However, the primary
   pathogenic determinants underlying TEC impairment and disease
   advancement in DKD progression remain unclear. This study reveals that
   asprosin (ASP) is up‐regulated and positively correlated with kidney
   dysfunction in DKD mice. Moreover, elevated ASP is mainly located in
   the renal TEC, and negatively impacts TEC. In addition,
   supraphysiological ASP concentration impairs mitochondrial dynamics and
   function in both DKD mice and HK2 cells. Mechanistically, ASP promotes
   Drp1 over‐SUMOylation, thus reducing Drp1 degradation and disrupting
   mitochondrial dynamics homeostasis. However, the mutation of
   Drp1‐SUMOylation modification sites alleviates the mitochondrial
   dynamics disorder, TEC injury, and phenotypic transformation induced by
   ASP. Also, it is further elucidated that such a regulatory effect of
   ASP on the Drp1‐SUMOylation modification is fulfilled by modulating
   PIAS1 or SENP1 (a de‐SUMOylation protease). Importantly, either adipose
   tissue‐specific ASP deficiency (ASP^−/−) or ASP antibody (AASP)
   intervention significantly alleviates the kidney injury and
   mitochondrial dynamics disorder induced by STZ/HFD. These findings
   identify ASP as a novel predictor of DKD and offer new therapeutic
   strategies for DKD prevention and treatment.

   Keywords: asprosin, diabetic kidney disease, mitochondrial dynamics,
   SUMOylation
     __________________________________________________________________

   ASP aggravates TEC injury and phenotypic transformation via
   mitochondrial dynamics disorder mediated by excessive Drp1 SUMOylation
   in DKD mice. However, either ASP deficiency or AASP effectively
   alleviates the kidney injury and mitochondrial dynamics disorder
   induced by STZ/HFD, highlighting that ASP could be an early diagnostic
   marker and potential therapeutic target of DKD.

   graphic file with name ADVS-12-e03259-g002.jpg

1. Introduction

   Diabetic kidney disease (DKD) is one of the major microvascular
   complications of diabetic patients and has become a major cause of
   end‐stage renal disease (ESRD) worldwide.^[ [40]^1 , [41]^2 ^] Renal
   tubular epithelial cells (TEC) are the main sites of glucose
   reabsorption, and their metabolic modes change with environmental
   changes.^[ [42]^3 ^] For example, under the glycotoxic condition, the
   metabolic mode of TEC switches from mitochondrial oxidative metabolism
   to glycolysis, which promotes the proliferation and differentiation of
   TEC.^[ [43]^4 ^] The process known as TEC epithelial‐mesenchymal
   transition (EMT), marked by epithelial cell polarity disruption and
   intercellular adhesion, along with the acquisition of mesenchymal
   characteristics, is widely acknowledged as a significant factor in the
   progression of DKD.^[ [44]^5 ^] It is worth noting that TEC phenotypic
   transformation plays a central role in the progression of renal
   tubulointerstitial fibrosis (RTF) and is an important pathological
   basis for ESRD. However, the current clinical diagnosis and treatment
   rationales have not been able to effectively prevent the development of
   ESRD.^[ [45]^6 ^] Therefore, it is urgent to ascertain the etiology and
   mechanism of DKD pathogenesis.

   It is commonly thought that re‐absorption is the predominant function
   of TEC, especially proximal tubules, and the process of active
   re‐absorption requires a large amount of energy. Thus, the proximal
   tubule is enriched with a large number of mitochondria due to its high
   energy demand and dependence on aerobic metabolism.^[ [46]^7 , [47]^8
   ^] Mitochondria are dynamic organelles that change to acclimate to the
   environment for survival and growth, including continuous fission and
   fusion.^[ [48]^9 ^] Clinical data show that mitochondrial fragmentation
   is increased in renal cortex tubular cells in the early stage of DKD
   patients.^[ [49]^10 ^] Disruptions of mitochondrial dynamics directly
   impact mitochondrial function, including proper biogenesis, autophagy,
   and redox state etc.^[ [50]^11 ^]

   It is well‐established that mitochondrial dynamics homeostasis is
   essential for maintaining the normal function of TEC. Among the
   numerous lines of evidence, dynamin‐related protein 1 (Drp1), a fission
   protein, plays a central role in altered dynamics, and its expression
   is increased in TEC of DKD mice. A key step in Drp1‐mediated
   mitochondrial fission is the translocation of Drp1 from the cytoplasm
   to the mitochondrial outer membrane (MOM).^[ [51]^12 ^] It is reported
   that post‐translational modification of Drp1 is involved in its
   translocation to MOM.^[ [52]^13 ^] Some studies show that Drp1
   SUMOylation promotes the association of Drp1 with MOM, and reduces its
   lysosomal degradation, thus further enhancing the mitochondrial
   fission.^[ [53]^14 , [54]^15 ^] Therefore, inhibiting the excessive
   modification of Drp1 by SUMO1 might be an important approach for
   maintaining the mitochondrial dynamics homeostasis and function in the
   progression of DKD.

   SUMOylation is a highly dynamic cascade that is catalyzed by the
   sequential enzymes, including E1 (SAE1/2), E2 (Ubc9), and E3 ligase in
   a manner analogous to ubiquitination, and it is also reversed by
   SUMO‐specific proteases, which cover six isoenzymes, including SENP1‐3
   and SENP5‐7.^[ [55]^12 ^] Protein inhibitors of activated STAT (PIAS)
   family proteins were originally identified as inhibitors of STAT, and
   they also possess E3 SUMO ligase property.^[ [56]^16 ^] Our previous
   study has shown that PIAS1 is involved in vascular endothelial insulin
   resistance induced by glucose and lipid metabolism disorders.^[ [57]^17
   ^] SENP1, as a SUMO‐specific protease, plays an important regulatory
   role in adipogenesis,^[ [58]^18 ^] glucose and lipid metabolism.^[
   [59]^19 , [60]^20 ^] These existing data suggest that PIAS1 and SENP1
   are involved in regulating the development of multiple metabolic
   diseases. However, it is unclear whether PIAS1 and SENP1 are involved
   in Drp1‐SUMO1 modification and regulate mitochondrial dynamics in the
   development of DKD.

   Asprosin (ASP), a peptide hormone mainly from white adipose tissue, was
   discovered in 2016 linked to a genetic mutation causing neonatal
   progeroid syndrome. Later studies show it affects appetite, metabolism,
   and heart health. High levels of ASP are linked to unhealthy habits,
   obesity, diabetes, and other metabolic disorders. Animal studies
   suggest that blocking ASP could improve metabolism, making it a
   potential treatment target.^[ [61]^21 , [62]^22 , [63]^23 ^] Our recent
   studies have demonstrated that ASP inhibits white adipose browning,^[
   [64]^24 ^] promotes vascular endothelial insulin resistance,^[ [65]^25
   ^] vascular endothelial inflammation,^[ [66]^26 ^] and aggravates
   metabolism‐associated fatty liver disease,^[ [67]^27 ^] suggesting that
   ASP is closely associated with the development of metabolic disorders.
   Several studies have shown that a supraphysiological concentration of
   ASP is an independent risk factor for the development and progression
   of DKD.^[ [68]^28 , [69]^29 , [70]^30 ^] However, the role of ASP in
   DKD progression remains largely unexplored. It has not been reported
   yet whether ASP is involved in renal tubular injury or by what
   mechanisms it works. Resolving the above issues is of great
   significance for early marker discovery and DKD therapy.

   In the present study, we indicate a positive correlation between ASP
   levels and renal function related indicators in DKD. In addition, we
   demonstrate that the regulation of the DKD process by ASP is realized
   primarily through promoting the excessive modification of Drp‐SUMO1,
   which leads to the disturbance of mitochondrial dynamics. In this
   regulation process, SENP1 and PIAS1 play important roles and would also
   be the potential targets. Altogether, these findings suggest that ASP
   could be an early diagnostic marker and a potential therapeutic target
   of DKD.

2. Results

2.1. ASP Intervention Causes or Exacerbates TEC Injury and Phenotypic
Transformation

   To investigate the link between ASP and DKD, we detected the serum ASP
   levels in DKD mice. We found that the levels of serum ASP were
   significantly increased starting with week 4 in DKD mice (P<0.001), and
   there was a gradually increased trend with the progression of DKD
   (Figure [71]S1a, Supporting Information); in addition, the levels of
   urinary ASP in DKD mice were much higher than those of ND mice (Figure
   [72]S1b, Supporting Information). Moreover, the ASP levels were
   positively correlated with the levels of serum creatinine (Scr) (Figure
   [73]S1c, Supporting Information) and blood urea nitrogen (BUN) (Figure
   [74]S1d, Supporting Information). To further investigate the role of
   ASP in DKD pathogenesis, ASP intervention was applied to the T2DM mice
   induced by STZ/HFD (Figure [75]1a). Our data showed that under the
   ND‐feeding, ASP intervention significantly elevated the Scr and BUN
   levels (Figure [76]1b,c), urinary ACRs levels (Figure [77]1d), and
   kidney weight/body weight ratio (KW/BW) (Figure [78]1e). Moreover,
   under the DKD, ASP intervention aggravated the elevations of the above
   parameters, suggesting that ASP intervention causes and worsens the DKD
   induced by STZ/HFD. Considering that ASP can regulate systemic
   glycolipid metabolism, we next observed the effects of ASP on
   glycolipid metabolism in mice. Our results revealed that ASP
   intervention caused and aggravated the glycolipid metabolism disorder
   and elevated systolic blood pressure (SBP) in DKD mice (Figure
   [79]S2a–m, Supporting Information), suggesting that the pro‐DKD effect
   of ASP may be due to in part inducing the glycolipid metabolism
   disorder.

Figure 1.

   Figure 1
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   ASP intervention causes or exacerbates TEC injury and phenotypic
   transformation. a) Schematic of the experimental design. Normal or
   STZ/HFD‐induced T2DM mice were treated with either normal saline (NS)
   or 0.5 µg· kg^−1· d^−1 ASP for 16 weeks. b,c) The levels of Scr and BUN
   (n = 6). d,e) The urinary albumin to creatinine ratios (ACRs) and
   kidney weight (KW)/body weight (BW) ratio in mice (n = 6). f,g)
   Co‐localization of ASP with different segment‐specific tubular markers
   of DKD kidney. ASP (red), aquaporin 1 (AQP1, marker of proximal renal
   tubular epithelial cell, green), Na^+‐Cl^− cotransporter (NCC, marker
   of distal renal tubular epithelial cell, green), DAPI (blue). Scale
   bar: original images, 50 µm; enlarged images, 20 µm. h–l)
   Representative images and quantitative analysis of (h, i, j)
   hematoxylin and eosin (H&E, black arrow: swelling, blue arrow:
   vacuolation), (h, k) periodic acid–Schiff (PAS) staining, and (h, l)
   Masson's trichrome staining of kidneys (n = 6). Scale bar: original
   images, 50 µm; enlarged images, 10 µm. m–q) Representative Western
   blotting and quantitative data showing the expressions of E‐Cadherin,
   Collagen III (Col III), Vimentin and α‐SMA of kidneys in various
   treatment groups. β‐actin was used as loading controls (n = 6). r–u)
   Representative images of immunohistochemical staining and quantitative
   data of (r, s) E‐Cadherin, (r, t) α‐SMA and (r, u) Collagen III (Col
   III) of kidneys after various treatment(n = 6). Scale bar: 20 µm. v–z)
   Representative Western blotting and quantitative data showing the
   expressions of E‐Cadherin, Collagen III (Col III), Vimentin and α‐SMA
   of HK2 cells under various treatment conditions. β‐actin was used as
   loading controls (n = 3). Data were presented as mean ± SEM.
   Statistical analysis was performed using the two‐way ANOVA followed by
   Tukey's multiple comparison test in all panels except for (a), (f),
   (h), (i), (m), (r) and (v). Non‐parametric Mann‐Whitney test was
   applied for statistical analysis of tubular injury scores (i). All
   tests were two tailed. ^* P<0.05, ^** P<0.01, ^*** P<0.001 versus ND+NS
   or Ctrl; ^# P<0.05, ^## P<0.01, ^### P<0.001 versus DKD+NS or HG.

   The renal tubule is the main site of glucose re‐absorption and it is
   the most vulnerable when it is subjected to high glucose stress. In
   order to further explore the direct impact and mechanism of ASP on the
   progression of DKD, we examined the ASP in situ expression in different
   segments of renal tubules by immunofluorescence. The data displayed
   that ASP was co‐located with proximal tubule marker aquaporin 1 (AQPI),
   but not with distal tubule marker Na^+‐Cl^− co‐transporter (NCC),
   indicating that ASP is mainly located in proximal renal tubule
   epithelial cells (Figure [81]1f,g) and it may play a significant role
   in the function of proximal tubules in DKD mice.

   We examined the impacts of ASP on TEC in DKD mice. Our morphological
   data showed that ASP caused TEC swelling (black arrow), vacuolation
   (blue arrow, H&E, Figure [82]1h,i), glomerular hypertrophy (H&E,
   Figure [83]1h,j), mesangial matrix expansion (PAS, Figure [84]1h,k) and
   collagen deposition (Masson, Figure [85]1h,l). Western blotting
   (Figure [86]1m–q) and immunohistochemistry (Figure [87]1r–u) revealed
   that ASP treatment resulted in and even exacerbated the TEC phenotypic
   transformation induced by DKD, evidenced as lower expression of
   E‐Cadherin and higher expression of Col III, Vimentin, and α‐SMA.
   Similar results were obtained from in vitro experiments
   (Figure [88]1v–z). Also, our in vitro data revealed that the promoting
   phenotypic transformation effect of ASP was concentration‐dependent
   (Figure [89]S3a–e, Supporting Information). Besides, the wound‐healing
   assay showed that ASP promoted or further enhanced the migration of HK2
   cells induced by HG (Figure [90]S4a,b, Supporting Information).
   Altogether, these data indicate that ASP intervention causes and even
   exacerbates the TEC injury and phenotypic transformation both in vivo
   and in vitro.

2.2. ASP Deficiency Inhibits TEC Injury and Phenotypic Transformation

   To further explore the role of ASP in DKD progression, an adipose
   tissue‐specific ASP‐deficient (ASP^−/−) mouse model was established
   (Figure [91]S5a–e, Supporting Information). Next, to evaluate the
   impact of ASP^−/− on the development of DKD, ASP^fl/fl and ASP^−/− mice
   were treated as Figure [92]2a. The results exhibited that ASP^−/−
   significantly alleviated the increased levels of Scr (Figure [93]2b),
   BUN (Figure [94]2c), urinary ACRs (Figure [95]2d), and KW/BW
   (Figure [96]2e) induced by DKD. In addition, ASP deficiency alleviated
   glucose and lipid metabolic disorder in DKD mice (Figure [97]S6a–m,
   Supporting Information), which suggests that ASP^−/− protects TEC in
   DKD mice by improving glucose and lipid metabolic disorder. Western
   blotting showed that E‐Cadherin was increased and Col III, Vimentin,
   and α‐SMA were decreased in ASP^−/−+DKD group compared with the DKD
   group (Figure [98]2f–j). Immunohistochemistry analysis also showed
   similar results (Figure [99]2k–n). The results of pathological staining
   showed that, the degree of renal tubular injury in ASP^−/−+ DKD group
   was reduced compared with DKD group, including the reductions of TEC
   swelling (black arrow), vacuolation (blue arrow, H&E,
   Figure [100]2o,p), glomerular hypertrophy (H&E, Figure [101]2o,q),
   mesangial matrix expansion (PAS, Figure [102]2o,r) and collagen
   deposition (Masson, Figure [103]2o,s). Besides, we detected serum FFA
   and adipokines, including leptin and adiponectin in DKD mice. The
   results showed that the disorders of adipokines occurred in DKD mice,
   and ASP deficiency significantly relieved the disorders in DKD mice
   (Figure [104]S7a–c, Supporting Information). These results suggest that
   ASP^−/− reverses the damage and phenotypic transition of TEC in the
   progression of DKD.

Figure 2.

   Figure 2
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   ASP deficiency inhibits TEC injury and phenotypic transformation. a)
   Schematic of the experimental design. b, c) The levels of Scr and BUN
   in mice (n = 6). d, e) The urinary ACRs and kidney weight (KW)/body
   weight (BW) ratio in mice (n = 6). f–j) Representative Western blotting
   and quantitative data showing the expressions of E‐Cadherin, Collagen
   III (Col III), Vimentin and α‐SMA in kidneys of ASP^fl/fl and ASP^−/−
   mice. β‐actin was used as a loading control (n = 6). k–n)
   Representative images of immunohistochemical staining and quantitative
   data of (k, l) E‐Cadherin, (k, m) α‐SMA, and (k, n) Collagen III (Col
   III) in kidneys of ASP^fl/fl and ASP^−/− mice (n = 6). Scale bar:
   20 µm. o‐s) Representative images and quantitative data of (o–q)
   hematoxylin and eosin (H&E, black arrow: swelling, blue arrow:
   vacuolation), (o, r) periodic acid–Schiff (PAS) staining, and (o, s)
   Masson's trichrome staining and quantitative of collagen content of
   kidneys (n = 6). Scale bar: original images, 50 µm; enlarged images,
   10 µm. t–x) Representative Western blotting and quantitative data
   showing the expressions of E‐Cadherin, Collagen III (Col III),
   Vimentin, and α‐SMA of HK2 cells. β‐actin was used as a loading control
   (n = 3). y) The co‐culture system of HK‐2 cells and 3T3‐L1 cells. z and
   aa) Representative images and quantitative data of the Transwell
   experiment of HK2 cells (n = 3). Scale bar: 50 µm. Data were presented
   as mean ± SEM. Statistical analysis was performed using the two‐way
   ANOVA followed by Tukey's multiple comparison test in all panels except
   for (a), (f), (k), (o), (p), (t), (y), and (z). The nonparametric
   Mann‐Whitney test was applied for statistical analysis of tubular
   injury scores (p). All tests were two tailed. ^** P<0.01, ^*** P<0.001
   versus ASP^fl/fl+ND or CM‐WT+Ctrl; ^# P<0.05, ^## P<0.01 versus
   ASP^fl/fl+DKD or CM‐WT+HG. ns, no significant difference versus
   ASP^fl/fl+ND or CM‐WT+Ctrl.

   To mimic the environment of adipose tissue‐specific ASP^−/− in vivo,
   HK2 cells were treated with the conditioned media (CM) from either
   WT‐3T3‐L1 adipocytes or ASP^−/−‐3T3‐L1 adipocytes. Western blotting
   data showed that the treatment with CM‐ASP^−/− markedly reversed the
   phenotypic transition induced by HG in HK2 cells, characterized as
   increased E‐Cadherin (Figure [106]2t,u), and decreased Col III
   (Figure [107]2t,v), Vimentin (Figure [108]2t,w) and ɑ‐SMA
   (Figure [109]2t,x). Another character is the enhancement of migration.
   To better ascertain the effects of ASP^−/− on migration of HK2 cells in
   vitro, a Transwell co‐culture system (0.8 µm) of 3T3‐L1 adipocytes and
   HK2 cells was established (Figure [110]2y). The results showed that ASP
   deficiency markedly reversed the elevated migration induced by HG
   (Figure [111]2z and aa). Taken together, these data suggest that
   ASP^−/− inhibits the DKD progression both in vivo and in vitro.

2.3. ASP Intervention Causes or Aggravates Mitochondrial Dysfunction by
Disturbing Mitochondrial Dynamics Homeostasis

   To further investigate the mechanism by which ASP promotes the
   progression of DKD, we performed the transcriptome analysis of the
   kidneys in ASP^fl/fl+DKD and ASP^−/−+DKD mice. KEGG pathway enrichment
   analysis showed that the differential expression genes (DEGs) were
   significantly enriched in the oxidative phosphorylation (OXPHOS),
   thermogenesis, citrate cycle (TCA cycle) pathway (Figure [112]3a),
   which were associated with mitochondrial metabolism and function. The
   heatmap showed that compared with the DKD mice, the gene expressions
   responsible for mitochondrial OXPHOS, thermogenesis, and TCA cycle were
   markedly elevated in the ASP^−/−+DKD mice (Figure [113]3b), suggesting
   that ASP^−/− promotes glycolipid metabolism by enhancing mitochondrial
   function in DKD mice. Mitochondrial dynamics homeostasis is
   indispensable for the maintenance of its function. Next, we evaluated
   the effects of ASP intervention on the mitochondrial dynamics of TEC.
   Western blotting data revealed that ASP intervention caused or
   exacerbated the mitochondrial dynamics disturbance in DKD mice,
   manifested as elevated Drp1 and Fis1 levels as well as decreased OPA1
   and MFN2 levels in mitochondria (Figure [114]3c–e). Numerous studies
   have shown that Drp1 plays a central role in mitochondrial fission.^[
   [115]^31 , [116]^32 ^] Therefore, here we focused primarily on the
   regulatory effects of ASP on Drp1. Immunofluorescence assay showed that
   ASP intervention promoted the co‐localization of Drp1 and Tomm20 (outer
   mitochondrial membrane marker), and the combined intervention of ASP
   and STZ/HFD further enhanced the levels of co‐localization
   (Figure [117]3f,g), indicating that ASP promotes Drp1 translocation
   from the cytosol to the mitochondria. In addition, we found that ASP
   increased the phosphorylation levels of Drp1 at s616 (Figure
   [118]S8a,b, Supporting Information). Moreover, ASP intervention
   significantly reduced ATP levels (Figure [119]3h) and increased
   production of mitochondrial ROS (Figure [120]3i,j). These data suggest
   that ASP results in mitochondrial dysfunction by disrupting the
   mitochondrial dynamics homeostasis in DKD mice.

Figure 3.

   ASP intervention causes or aggravates mitochondrial dysfunction by
   disturbing mitochondrial dynamics homeostasis. a) Analysis of KEGG
   enrichment pathways for differentially expressed genes (DEGs) in the
   kidneys of DKD and DKD+ASP^−/− mice (n = 3). b) Heatmap of DEGs related
   to mitochondrial function in the kidneys of ASP^fl/fl+DKD and
   ASP^−/−+DKD mice (n = 3). c–e) Representative Western blotting and
   quantitative data showing the expressions of OPA1, MFN2, Drp1, and Fis1
   in the cytosolic fraction and mitochondrial fraction of renal tissue.
   β‐actin was used as a marker for the cytoplasmic fraction, and COX IV
   was used as a marker for the mitochondrial fraction (n = 6). f, g)
   Co‐localization and its analysis of Drp1 with Tomm20 in kidneys (n =
   6). Drp1 (red), Tomm20 (green), DAPI (blue). Scale bar: original
   images, 50 µm; enlarged images, 20 µm. h) ATP levels in kidneys (n =
   6). i, j) Representative images of MitoSOX (red) and its quantitative
   data in kidneys (n = 6). Scale bar: 50 µm. k, l) Representative images
   of MitoTraker (red) and its quantitative data in HK2 cells (n = 3).
   Scale bar: original images, 10 µm; enlarged images, 5 µm. m, n)
   Representative images of transmission electron microscope (TEM) and
   quantitative data in HK2 cells (n = 3). Scale bar: original images,
   500 nm; enlarged images, 200 nm. o–q) Representative Western blotting
   and quantitative data showing the expressions of OPA1, MFN2, Drp1, and
   Fis1 in the cytosolic fraction and mitochondrial fraction of HK2 cells.
   β‐actin and COX IV were used as loading controls (n = 3). r, s)
   Co‐localization of Drp1 with MitoTraker and quantitative data in HK2
   cells (n = 3). MitoTraker (red), Drp1 (green), DAPI (blue). Scale bar:
   original images, 20 µm; enlarged images, 5 µm. t) ATP levels in HK2
   cells (n = 3). u, v) Detection and quantification of Mito‐SOX in HK2 by
   Flow cytometry. w, x) Representative images of JC‐1 staining and its
   quantitative data in HK2 cells (n = 3). Scale bar: 50 µm. Data were
   presented as mean ± SEM. Statistical analysis was performed using
   one‐way (d, e, p, q, v) or two‐way ANOVA (g, h, j, l, n, s, t, x)
   followed by Tukey's multiple comparison test. All tests were two
   tailed. ^* P<0.05, ^** P<0.01, ^*** P<0.001 versus ND+NS or Ctrl; ^#
   P<0.05, ^## P<0.01, ^### P<0.01 versus DKD or HG.

   graphic file with name ADVS-12-e03259-g011.jpg

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   Subsequently, Mito‐Tracker and transmission electron microscope (TEM)
   were used to examine the morphology of mitochondria in HK2 cells. Our
   results showed that ASP promoted or exacerbated mitochondrial
   fragmentation induced by HG (Figure [122]3k–n). Moreover, the
   expressions of mitochondrial dynamics related proteins were similar to
   those in vivo experiments (Figure [123]3o–q). Also, the results of
   immunofluorescence showed that ASP promoted the Drp1 expression induced
   by HG in mitochondria (Figure [124]3r,s). These results suggest that
   ASP may promote mitochondrial fission in HK2 cells. Meanwhile, ASP
   decreased ATP production (Figure [125]3t), increased mitochondrial ROS
   levels (Figure [126]3u,v), and promoted the conversion of JC‐1 polymers
   to monomers (mitochondrial membrane potential decreased,
   Figure [127]3w,x), demonstrating that ASP intervention alone impairs
   mitochondrial function, and ASP+HG intervention further aggravates the
   mitochondrial dysfunction. These data suggest that ASP results in
   mitochondrial dysfunction by disrupting the mitochondrial dynamics
   homeostasis in HK2 cells.

2.4. ASP Deficiency Alleviates Mitochondrial Dysfunction by Restoring
Mitochondrial Dynamics Homeostasis

   We have found that ASP intervention impaired mitochondrial dynamics and
   function in the kidneys of DKD mice. Subsequently, we explored the
   effects of ASP deficiency on mitochondrial dynamics and function. The
   results showed that compared with DKD group, the expressions of
   mitochondrial fission proteins Drp1 and Fis1 in mitochondria were
   decreased, while the expressions of fusion proteins OPA1 and MFN2 in
   mitochondria were increased in the kidneys of ASP^−/−+DKD mice (Figure
   [128]4a–c). To further explore the effects of ASP^−/− on Drp1, Western
   blotting and immunofluorescence co‐localization assay were performed.
   We found that ASP^−/− significantly reduced the expression levels of
   p‐Drp1 s616 in the kidneys of DKD mice (Figure [129]4d,e), and
   decreased the co‐localization of Drp1 and Tomm20 (Figure [130]4f,g).
   These data suggest that ASP^−/− alleviates the mitochondrial dynamics
   disorder in DKD mice. At the same time, we detected the indicators
   related to mitochondrial function, and the results revealed that
   ASP^−/− significantly reduced mitochondrial ROS levels
   (Figure [131]4h,i) and increased ATP levels (Figure [132]4j) in the
   kidneys of DKD mice. These results indicate that ASP^−/− has protective
   effects on mitochondrial function in the kidneys of DKD mice.

Figure 4.

   Figure 4
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   ASP deficiency alleviates mitochondrial dysfunction by restoring
   mitochondrial dynamics homeostasis. a–c) Representative Western
   blotting and quantitative data showing the expressions of OPA1, MFN2,
   Drp1, and Fis1 in cytosolic fraction and mitochondrial fraction in
   kidneys. β‐actin was used as a marker for the cytoplasmic fraction, and
   COX IV was used as a marker for the mitochondrial fraction (n = 6). d,
   e) Representative Western blotting and quantitative data showing the
   expressions of p‐Drp1 s616 and Drp1 in kidneys. β‐actin were used as
   loading controls (n = 6). f, g) Co‐localization of Drp1 with Tomm20 and
   its quantitative data in kidneys (n = 6). Drp1 (red), Tomm20 (green),
   DAPI (blue). Scale bar: original images, 50 µm; enlarged images, 20 µm.
   h, i) Representative images of MitoSOX (red) and its quantitative data
   in kidneys (n = 6). Scale bar: 50 µm. j) ATP levels in kidneys (n = 6).
   Data were presented as mean ± SEM. Statistical analysis was performed
   using one‐way (b, c) or two‐way ANOVA (e, i, j, g) followed by Tukey's
   multiple comparison test. All tests were two tailed. ^** P < 0.01, ^***
   P < 0.001 versus ASP^fl/fl+ND; ^# P < 0.05, ^## P < 0.01 versus
   ASP^fl/fl+DKD. ns, no significant difference versus ASP^fl/fl+ND.

2.5. Pro‐Injury and Phenotypic Transformation Effects of ASP are Mediated by
Drp1

   Next, we investigated the underlying mechanisms by which ASP induces
   TEC injury and phenotypic transformation. Drp1 serves as an important
   protein regulating mitochondrial dynamics, and it remains uncertain
   whether the impaired effects of ASP on TEC would be mediated by Drp1 in
   DKD mice. To address it, we knocked down Drp1 (Figure [134]S9a–c,
   Supporting Information) and utilized pharmacologic inhibitors of Drp1
   in HK2 cells. Our results displayed that down‐regulation of Drp1
   markedly antagonized the ASP‐mediated TEC injury and phenotypic
   transformation, especially under the HG, evidenced as increased
   E‐Cadherin, and decreased Vimentin, ɑ‐SMA as well as Col III (Figure
   [135]5a–f). Immunofluorescence data also showed that Drp1
   down‐regulation significantly reversed the decrease of E‐Cadherin
   (Figure [136]5g,i) and the increase of α‐SMA (Figure [137]5h,j)
   triggered by ASP. In addition, Midivi‐1, a Drp1 inhibitor,
   significantly reversed the TEC phenotypic transformation induced by ASP
   in HK2 cells, and the inhibitory effect of Midivi‐1 was
   concentration‐dependent (Figure [138]5k,l). These dates indicate that
   the pro‐injury and phenotypic transformation effects of ASP are
   mediated by Drp1.

Figure 5.

   Figure 5
   [139]Open in a new tab

   Pro‐injury and phenotypic transformation effects of ASP are mediated by
   Drp1. a–f) Representative Western blotting and quantitative data
   showing the expressions of E‐Cadherin, Collagen III (Col III),
   Vimentin, α‐SMA, and Drp1 in HK2 cells after Drp1 shRNA was transfected
   to the HK2 cells. β‐actin was used as a loading control (n = 3). g–j)
   Representative images of immunofluorescence staining and quantitative
   data of E‐Cadherin, α‐SMA in HK2 cells (n = 3). Scale bar: 100 µm. k,
   l) Representative Western blotting and quantitative data showing the
   expressions of E‐Cadherin, Collagen III (Col III), Vimentin, and α‐SMA
   in HK2 cells. β‐actin was used as a loading control (n = 3). Data were
   presented as mean ± SEM. Statistical analysis was performed using
   one‐way (l) or two‐way ANOVA (b‐f, i, j) followed by Tukey's multiple
   comparison test. All tests were two tailed. ^* P < 0.05, ^** P < 0.01,
   ^*** P<0.001 versus Vehicle+Ctrl or HG+Ctrl; ^# P < 0.05, ^## P < 0.01,
   ^### P < 0.001 versus Vehicle+ASP or HG+ASP; ^$ P < 0.05, ^$$ P < 0.01
   versus Vehicle+HG; ^& P < 0.05, ^&& P < 0.01,^&&& P < 0.001 versus
   Vehicle+HG+ASP.

2.6. ASP Intervention Enhances Drp1‐SUMO1 Modification

   It is reported that Drp1 SUMOylation stabilizes its conformation and
   influences its localization and activity. Over‐modification of Drp1 by
   SUMO1 causes excessive accumulation of Drp1 in OMM, leading to abnormal
   mitochondrial fission.^[ [140]^33 ^] Our results exhibited that ASP
   intervention significantly elevated SUMO1 levels, which were consistent
   with the elevated levels of Drp1 in mitochondria. Furthermore, the
   combined intervention of ASP with STZ/HFD further elevated the levels
   of SUMO1 (Figure [141]6a,b). Co‐IP results further demonstrated that
   ASP intervention, under the condition of whether ND or DKD markedly
   increased the levels of Drp1‐SUMO1 modification (Figure [142]6c,d),
   suggesting that ASP intervention promotes Drp1 SUMOylation. Besides, we
   investigated the in situ expressions and co‐localization of Drp1 and
   SUMO1 in the kidneys of DKD mice by immunofluorescence. The results
   revealed that ASP promoted the in situ expression and co‐localization
   of Drp1 and SUMO1 (Figure [143]6e,f). Similar results were obtained
   from the in vitro data (Figure [144]6g–j). Taken together, these data
   suggest that ASP intervention enhances Drp1 SUMOylation modification in
   vivo and in vitro. In addition, we examined the effects of ASP^−/− on
   the modification of Drp1‐SUMO1 in the kidneys of DKD mice. The results
   showed that ASP^−/− significantly reduced the levels of Drp1‐SUMO1
   (Figure [145]S10a,b, Supporting Information) and the co‐location of
   Drp1 and SUMO1 (Figure [146]S10c,d, Supporting Information).

Figure 6.

   Figure 6
   [147]Open in a new tab

   ASP intervention enhances Drp1‐SUMO1 modification. a, b) Representative
   Western blotting and quantitative data showing the expression of SUMO1
   in the mitochondrial fraction in kidneys. COX IV was used as loading
   controls (n = 6). c, d) SUMOylation of Drp1 levels detected by Co‐IP in
   kidneys (n = 6). e, f) Co‐localization of Drp1 with SUMO1 and its
   quantitative data in kidneys (n = 6). Drp1 (red), SUMO1 (green), DAPI
   (blue). Scale bar: original images, 50 µm; enlarged images, 20 µm. g,
   h) SUMOylation of Drp1 levels detected by Co‐IP in HK2 cells (n = 3).
   i, j) Co‐localization of Drp1 with SUMO1 and its quantitative data in
   HK2 cells (n = 3). Drp1 (red), SUMO1 (green), DAPI (blue). Scale bar:
   original images, 20 µm; enlarged images, 5 µm. Data were presented as
   mean ± SEM. Statistical analysis was performed using two‐way ANOVA
   followed by Tukey's multiple comparison test (b, f, d, j, h). All tests
   were two tailed. ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001 versus ND+NS
   or Ctrl; ^# P < 0.05, ^## P < 0.01 versus DKD+NS or HG.

2.7. Drp1‐SUMOylation Site Mutation Alleviates TEC Mitochondrial Dysfunction
and Phenotypic Transformation Induced by ASP

   It is reported that Drp1 contains GTPase domain, a Middle domain, a
   Variable domain, and GTPase‐effector domain (GED) and the SUMOylation
   of Drp1 (NM_0 05690.4) occurs at K557, K560, K569, and K571 sites in
   the Variable domain.^[ [148]^34 ^] To further elucidate the roles of
   Drp1 SUMOylation in TEC injury and phenotypic transformation induced by
   ASP, a mutation of Drp1 SUMOylation sites at K557, K560, K569, and
   K571, abbreviated as Drp1‐4KR was constructed (Figure [149]7a). Our
   results showed that Drp1‐4KR mutant notably reduced the Drp1‐SUMO1
   levels compared to Drp1‐WT (Figure [150]7b,c) and alleviated the
   mitochondrial dynamics disorder induced by ASP, mainly manifested as
   the increased expressions of mitochondrial fusion proteins OPA1 and
   MFN2 and decreased expressions of mitochondrial fission proteins Drp1
   and Fis1 (Figure [151]7d–f) and mitochondrial fragmentation
   (Figure [152]7g–j). Moreover, Drp1‐4KR alleviated the impairment of
   mitochondrial function caused by ASP in HK2, mainly indicated as the
   elevated ATP levels (Figure [153]7k). Also, Drp1‐4KR significantly
   reversed the TEC phenotypic transformation induced by ASP, evidenced as
   the increased epithelial marker E‐Cadherin and the decreased
   mesenchymal markers Col III, Vimentin, and α‐SMA (Figure [154]7l–p).
   Overall, these data indicate that Drp1 SUMOylation site mutation
   mitigates the mitochondrial dysfunction and phenotypic transformation
   of TEC induced by ASP.

Figure 7.

   Figure 7
   [155]Open in a new tab

   Drp1‐SUMOylation site mutation alleviates TEC mitochondrial dysfunction
   and phenotypic transformation induced by ASP. a) Domain structures of
   wild‐type Drp1 (Drp1‐WT) and a non‐SUMOylatable Drp1 mutant (Drp1‐4KR).
   A D‐octadecapeptide sequence, comprising 18 amino acid residues
   (554–571) encompassing in the Variable domain. Drp1‐4KR was created by
   substituting four lysine residues (K, red) which are SUMOylation sites
   in the D‐octadecapeptide with arginine residues (R). b, c) SUMOylation
   of Drp1 levels detected by Co‐IP of HK2 cells in Flag‐Drp1‐4KR groups
   (n = 3). d–f) Representative Western blotting and quantitative data
   showing the expressions of OPA1, MFN2, Flag‐Drp1 and Fis1 in cytosolic
   fraction and mitochondrial fraction of HK2 cells. β‐actin was used as a
   marker for the cytoplasmic fraction, and COX IV was used as a marker
   for the mitochondrial fraction (n = 3). g, h) Representative images of
   MitoTraker (red) and its quantitative data in HK2 cells (n = 3). Scale
   bar: original images, 10 µm; enlarged images, 5 µm. i, j)
   Representative images of TEM and quantitative data in HK2 cells (n =
   3). Scale bar: original images, 500 nm; enlarged images, 200 nm. k) ATP
   levels in HK2 cells (n = 3). l–p) Representative Western blotting and
   quantitative data showing the expressions of E‐Cadherin, Collagen III
   (Col III), Vimentin, and α‐SMA in HK2 cells. β‐actin was used as a
   loading control (n = 3). Data were presented as mean ± SEM. Statistical
   analysis was performed using the unpaired Student's t‐test (c), one‐way
   (e, f) or two‐way ANOVA (h–k, m–p) followed by Tukey's multiple
   comparison test. All tests were two tailed. ^* P < 0.05, ^** P < 0.01,
   ^*** P < 0.001 versus Drp1‐WT+Ctrl; ^# P < 0.05, ^## P < 0.01 versus
   Drp1‐WT+ASP; ^$$ P < 0.01 versus Drp1‐WT+HG; ^&& P < 0.001 versus
   Drp1‐WT+HG+ASP.

2.8. ASP Pro‐TEC Injury and Phenotypic Transformation Mediated by Drp1‐SUMO1
is Regulated by SENP1 and PIAS1

   PIAS1 SUMOylates substrate proteins as E3 ligase of SUMOylation while
   SENP1 de‐SUMOylates them as a de‐SUMOylase. Therefore, they determine
   the substrate‐SUMO1 levels. As ASP promotes the TEC injury and
   phenotypic transformation by elevating Drp1‐SUMO1 levels, it remains
   unclear whether ASP would affect PIAS1 and SENP1. Hence, we detected
   the expressions of PIAS1, SENP1, and ASP in the kidneys of DKD mice. We
   found that the expressions of PIAS1 and ASP were elevated in a positive
   correlation manner (Figure [156]8a,c), while the expressions of SENP1
   and ASP embodied a negative correlation (Figure [157]8b,d). Similarly,
   ASP intervention notably increased the expression of PIAS1 and
   decreased the expression of SENP1 in the kidneys of DKD mice
   (Figure [158]8e–g). Subsequently, we performed the rescue experiments
   by PIAS1 knockdown and SENP1 over‐expression in HK2 cells. The results
   revealed that both PIAS1 knockdown (Figure [159]S11a,b, Supporting
   Information) and SENP1 over‐expression (Figure [160]S11c,d, Supporting
   Information) significantly reduced the Drp1‐SUMO1 levels. More
   importantly, both PIAS1 knockdown (Figure [161]8h–l) and SENP1
   over‐expression (Figure [162]8m–q) reversed the phenotypic
   transformation induced by ASP in HK2 cells, characterized by increased
   E‐Cadherin and decreased Col III, Vimentin and α‐SMA, indicating that
   ASP promotes the TEC phenotypic transformation by up‐regulating the
   expression of PIAS1 and down‐regulating the expression of SENP1, and
   also suggesting that PIAS1 and SENP1 would be therapeutic targets of
   DKD. Subsequently, it was still unclear whether ASP would interact with
   PIAS1 or SENP1. Molecular docking results showed that ASP and SENP1 had
   a strong combined energy (ΔiG = −12.5 kcal mol^−1, Figure [163]8r–u),
   which was further verified by Co‐IP results in the kidneys of DKD mice
   (Figure [164]8v). However, to our surprise, there was no physical
   interaction between ASP and PIAS1, suggesting that ASP affects PIAS1,
   maybe through other manners, which needs to be further explored.

Figure 8.

   Figure 8
   [165]Open in a new tab

   ASP pro‐TEC injury and phenotypic transformation mediated by Drp1‐SUMO1
   is regulated by SENP1 and PIAS1. a) Representative images of
   immunofluorescence staining of ASP and PIAS1 in kidneys. ASP (red),
   PIAS1 (green), and DAPI (blue). Scale bar: 50 µm. n = 6. b)
   Representative images of immunofluorescence staining of ASP and SENP1
   in kidneys. ASP (red), SENP1 (green), and DAPI (blue). Scale bar:
   50 µm. c) Correlation between ASP and PIAS1 in DKD mice (n = 36). d)
   Correlation between the ASP and SENP1 in DKD mice (n = 36). e–g)
   Representative Western blotting and quantitative data showing the
   expression of PIAS1 and SENP1 in HK2 cells. β‐actin was used as a
   loading control. h–q) Representative Western blotting and quantitative
   data showing the expressions of E‐Cadherin, Collagen III (Col III),
   Vimentin, and α‐SMA of HK2 cells. β‐actin was used as a loading
   control. r) The 3D structure of ASP was predicted using the SWISS‐MODEL
   database. s) The 3D structures of SENP1 were obtained from the PDB
   database. t, u) Molecular docking demonstrated the interaction between
   ASP (red) and SENP1 (blue) (ΔiG = ‐12.5 kcal mol^−1), which was
   obtained through the HADOCK molecular docking platform. v) Interaction
   between ASP and PIAS1 or SENP1 detected by Co‐IP in the kidneys of ND
   and DKD mice. Data were presented as mean ± SEM. Statistical analysis
   was performed using the two‐way ANOVA followed by Tukey's multiple
   comparison test (f, g, i‐l, n‐q). Pearson correlation was used to
   calculate the correlation coefficient (c, d). All tests were two
   tailed. ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001 versus ND+NS or
   Vehicle+Ctrl; ^P < 0.05, ^^P < 0.01 versus DKD+NS; ^# P < 0.05, ^## P <
   0.01 versus Vehicle+ASP; ^$ P < 0.05, ^$$ P < 0.01, ^$$$ P < 0.001
   versus Vehicle+HG; ^&& P < 0.001 versus Vehicle+HG+ASP.

2.9. Neutralization of ASP Alleviates TEC Injury and Phenotypic
Transformation in DKD Mice

   The above studies suggest that ASP may be a promising target for the
   treatment of DKD. ASP is a newly discovered adipokine, and up to date
   there are no drugs that can combat ASP. Herein, an ASP neutralization
   antibody (AASP) was utilized to evaluate whether it can resist the
   negative effects of ASP on TEC. DKD mice induced by STZ/HFD were
   treated with AASP (Figure [166]9a). We found that AASP treatment
   markedly reduced the levels of Scr, BUN, urinary ACRs and KW/BW ratio
   compared with DKD mice (Figure [167]9b–e). Further research showed that
   AASP significantly reduced the content of mitochondrial ROS
   (Figure [168]9f,g), increased the levels of ATP (Figure [169]9h), and
   improved the mitochondrial dynamics homeostasis (Figure [170]9i–k) in
   kidneys of DKD mice. These data indicate that AASP has protective
   effects on mitochondrial dynamics and function of kidneys in DKD mice.
   Moreover, H&E staining results displayed that AASP reduced TEC swelling
   (black arrow), vacuolation (blue arrow, Figure [171]9l,m), and
   glomerular hypertrophy (Figure [172]9l,n). Also, PAS and Masson
   staining data exhibited that AASP alleviated mesangial matrix expansion
   (Figure [173]9l,o) and collagen deposition (Figure [174]9l,p). The
   results of Western blotting (Figure [175]9q,r) and immunohistochemistry
   (Figure [176]9s–v) showed that AASP inhibited the progress of TEC
   phenotypic transformation, evidenced by increasing E‐Cadherin and
   decreasing the expressions of Col III, Vimentin, and α‐SMA. Altogether,
   these results demonstrate that AASP alleviates TEC injury and
   phenotypic transformation as well as mitochondrial dysfunction in DKD
   mice.

Figure 9.

   Figure 9
   [177]Open in a new tab

   Neutralization of ASP alleviates TEC injury and phenotypic
   transformation in DKD mice. a) Schematic of the experimental design.
   STZ/HFD‐induced T2DM mice were treated with IgG or 0.5 µg · kg^−1 ·
   d^−1 ASP neutralizing antibody (AASP) for 16 weeks. b, c) The levels of
   Scr and BUN levels in mice (n = 6). d, e) The urine albumin to
   creatinine ratios (Urinary ACRs) and kidney weight (KW)/body weight
   (BW) ratio in mice (n = 6). f, g) Representative images of MitoSOX
   (red) and its quantitative data in kidneys (n = 6). Scale bar: 50 µm.
   h) ATP levels in kidneys (n = 6). i–k) Representative Western blotting
   and quantitative data showing the expressions of OPA1, MFN2, Drp1 and
   Fis1 in cytosolic fraction and mitochondrial fraction of kidneys.
   β‐actin was used as a marker for the cytoplasmic fraction, and COX IV
   was used as a marker for the mitochondrial fraction (n = 6). l–p)
   Representative images and quantitative data of (l–n) hematoxylin and
   eosin (H&E, black arrow: swelling, blue arrow: vacuolation), (l, o)
   periodic acid–Schiff (PAS), and (l, p) Masson's trichrome staining in
   kidneys (n = 6). Scale bar: original images, 50 µm; enlarged images,
   10 µm. q, r) Representative Western blotting and quantitative data
   showing the expressions of E‐Cadherin, Collagen III (Col III),
   Vimentin, and α‐SMA in kidneys. β‐actin was used as a loading control
   (n = 6). s–v) Representative images of immunohistochemical staining and
   quantitative data of E‐Cadherin, α‐SMA, and Collagen III (Col III) in
   kidneys (n = 6). Scale bar: 20 µm. Data were presented as mean ± SEM.
   Statistical analysis was performed using the one‐way ANOVA followed by
   Tukey's multiple comparison test in all panels except for (a), (f),
   (i), (l), (m), (q), and (s). The nonparametric Mann‐Whitney test was
   applied for statistical analysis of tubular injury scores (m). All
   tests were two tailed. ^** P < 0.01, ^*** P < 0.001 versus ND; ^# P <
   0.05, ^## P < 0.01, ^### P < 0.01 versus DKD.

3. Discussion

   DKD affects ≈40% of diabetic patients, and ≈20%–40% of those with DKD
   will develop to ESRD.^[ [178]^35 ^] Once renal structural damage
   occurs, it is exceedingly difficult to reverse the damage that has
   already taken place.^[ [179]^36 , [180]^37 , [181]^38 ^] TEC injury
   occurs in the early stage of ESRD and is regulated by hyperglycemia,
   transforming growth factor beta 1 (TGF‐β), interleukin 6 (IL‐6), and
   other factors.^[ [182]^39 , [183]^40 ^] Here, we found that the levels
   of circulating ASP were positively correlated with the levels of BUN
   and Scr, and the levels of urinal ASP were significantly elevated in
   the DKD mice. Therefore, we for the first time propose that ASP might
   be a novel renal injury factor.

   As a newly discovered adipokine, ASP levels were found elevated in the
   kidneys in patients with DKD.^[ [184]^29 ^] The proximal tubule plays a
   key role in maintaining metabolic homeostasis in the body through the
   reabsorption of nutrients (glucose and amino acids). ASP is primarily
   involved in the regulation of glucose metabolism and energy
   homeostasis. Our present results also demonstrate that ASP is involved
   in the development of DKD by interacting with the TEC. Our results show
   that ASP is abundantly accumulated in the proximal tubules and
   seriously damages the structure and function of the renal tubules in
   DKD mice. It is reported that tubular damage initiates TEC phenotypic
   transformation, including epithelial marker loss and mesenchymal marker
   acquisition, primarily representing a maladaptive response to injury
   that accelerates fibrosis.^[ [185]^35 ^] We found that ASP decreased
   the expression of epithelial markers (E‐Cadherin), while increasing the
   expression of mesenchymal markers (Col III, Vimentin, and α‐SMA) in
   mouse kidneys and HK2 cells. More importantly, ASP intervention results
   in TEC mitochondrial dysfunction, which further causes glycolipid
   metabolic disorder and TEC damage. Our data have shown that ASP
   intervention exacerbates the glucose and lipid metabolic disorder in
   DKD mice, which is mainly attributed to TEC mitochondrial dysfunction
   induced by ASP. As previously reported, we constructed adipose
   tissue‐specific ASP‐deficient mice using CRISPR‐Cas9 technology.^[
   [186]^24 , [187]^25 , [188]^41 ^] We found that ASP deficiency
   significantly reversed TEC phenotypic transformation and ameliorated
   the mitochondrial function. In addition, to explore whether ASP^−/−
   affects other adipokine levels, we tested the levels of serum leptin,
   adiponection, and FFA. Our results suggest that ASP^−/− may be combined
   with other adipokines to regulate the DKD process. Therefore, we
   conclude that ASP aggravates the TEC injury and phenotypic
   transformation in DKD mice, which deserves our further attention on its
   mechanisms.

   Mitochondria are the primary organelles responsible for energy
   metabolism in the kidney regardless of whether the health or DKD
   status.^[ [189]^7 , [190]^42 , [191]^43 ^] Renal re‐absorption mainly
   occurs in the proximal tubule, which is enriched in mitochondria due to
   its high energy demand.^[ [192]^44 , [193]^45 , [194]^46 ^]
   Mitochondrial dynamics homeostasis is necessary for maintaining normal
   mitochondrial function. Our previous studies found that ASP disturbed
   the mitochondrial dynamics mediated by Drp1 and caused vascular
   endothelial cell dysfunction.^[ [195]^25 ^] In order to clarify whether
   the effects of ASP on TEC would be related to the mitochondrial
   dynamics, we focused on the regulatory roles of ASP on Drp1. We found
   that ASP promoted the aggregation of Drp1 on mitochondria and disrupted
   mitochondrial dynamics homeostasis, including the reduced expression of
   fusion proteins OPA1 and MFN2, increased expression of fission proteins
   Drp1 and Fis1, and elevated mitochondrial fragmentation in the kidneys
   of both normal and DKD mice. Similar results were obtained from the in
   vitro experiments. Mitochondrial dynamics disturbance often results in
   mitochondrial dysfunction. Our results revealed that ASP intervention
   also caused mitochondrial dysfunction, such as increased mitochondrial
   ROS levels, decreased ATP levels, and mitochondrial membrane potential
   (decreased JC‐1 aggregates). In contrast, ASP deficiency significantly
   reversed the impairments of mitochondrial dynamics and function by ASP.

   Our results indicate that Drp1 plays an important role in the process
   of mitochondrial dynamics disorder induced by ASP. As expected, both
   Drp1 knockdown and intervention with Midivi‐1 (a Drp1 inhibitor) in HK2
   cells significantly reversed the disruptive effects of ASP on
   mitochondrial dynamics and TEC injury and phenotypic transformation. It
   is reported that post‐translational modification of Drp1 affects its
   translocation from the cytosol to the mitochondria and activity. It is
   well established that Drp1 SUMOylation can greatly stabilize Drp1
   conformation and inhibit its degradation by lysosomes.^[ [196]^14 ,
   [197]^47 ^] Our study showed that Drp1‐SUMO1 modification was enhanced
   in the kidneys of DKD mice, and ASP intervention further enhanced the
   levels of Drp1‐SUMO1. On the contrary, we mutated the SUMO modification
   sites of Drp1 and found that the disturbance of mitochondrial dynamics
   and TEC injury, and phenotypic transformation induced by ASP in HK2
   cells were greatly abolished. Altogether, these results suggest that
   Drp1‐SUMO1 over‐modification plays an important role in the process of
   TEC injury and phenotypic transformation induced by ASP.

   PIAS1 and SENP1 are E3‐ubiquitin ligase and SUMO‐specific protease,
   respectively, mediating protein SUMO modification and de‐SUMO
   modification.^[ [198]^48 ^] Regulation of PIAS1 and SENP1 determines
   the SUMO modification levels of substrate proteins. Our data showed
   that PIAS1 and SENP1 also mediated Drp1‐SUMO1 modification in DKD. To
   our surprise, the expression level of ASP was positively correlated
   with that of PIAS1, but negatively correlated with that of SENP1. In
   addition, we found that both PIAS1 knockdown and SENP1 over‐expression
   reversed the damaged effects of ASP on HK2 cells. However, the
   molecular docking results showed that ASP can interact with SENP1, and
   the results of Co‐IP further verified the interaction between ASP and
   SENP1 rather than PIAS1. Therefore, it is rational that PIAS1 and SENP1
   would be potential therapeutic targets for treating DKD. Excitingly, we
   found that AASP, an antibody to ASP, mitigated the TEC injury and
   phenotypic transformation, rescued the mitochondrial dynamics
   homeostasis, reduced mitochondrial ROSand increased ATP levels,
   suggesting that the negative regulation for ASP would be a therapeutic
   strategy for DKD.

   However, there are still some limitations and unsolved issues. First,
   this study primarily focused on the injury effects of ASP on renal
   tubules, while paying less attention to glomeruli. Second, we only
   investigated the effects of Drp1‐SUMO1 modification on DKD induced by
   ASP, and did not confirm which SUMO modification site could play a
   major role in this process. Finally, although several studies have
   shown that the receptors of ASP in the central nervous system and liver
   are olfr734^[ [199]^49 ^] and Ptprd,^[ [200]^50 ^] respectively, what
   ASP receptor would work and whether the interaction between ASP and
   SENP1 would be mediated through membrane receptors of TEC need to be
   further investigated. Addressing these issues is of great significance
   for suppressing the negative effects of ASP on TEC.

   In conclusion, we found that ASP intervention damages TEC and
   mitochondrial dynamic homeostasis in DKD mice and HK2 cells. In
   contrast, either ASP deficiency or AASP significantly alleviates the
   TEC injury and phenotypic transformation in DKD mice. Mechanistically,
   ASP causes and even aggravates TEC injury and phenotypic transformation
   by promoting excessive Drp1‐SUMO1 modification, in which PIAS1 and
   SENP1 are highly correlated with ASP and play important regulatory
   roles (Figure 10). The findings indicate that ASP would be an early
   marker of DKD and an important target of DKD treatment.

4. Experimental Section

Experimental Animals

   Animal (Nanfang Model Biotechnology, Shanghai, CHN) experiments were
   approved by the Laboratory Animal Ethic and Welfare Committee of
   Nanchang University (approval No. NCULAE‐20241213001) and conducted in
   accordance with the National Research Council Guide for the Care and
   Use of Laboratory Animals (8th Edition, 2011, Washington, DC published
   by the National Academies Press). The animals were housed in standard
   cages under controlled conditions of temperature (23 ± 1 °C) and
   humidity (50–60%), with a 12‐h light/dark cycle, and had ad libitum
   access to water. Adipose tissue‐specific ASP‐deficient mice were
   generated as previously described.^[ [201]^24 , [202]^25 ^] Random
   allocation of groups was conducted using a computer‐based random order
   generator.

Mouse Type 2 Diabetes Mellitus (T2DM) Models

   Mouse T2DM model was established as previously described by Xie,
   et al.^[ [203]^51 ^] Briefly, after 7 weeks of high‐fat diet (HFD)
   feeding, ASP^−/− mice and ASP^fl/fl littermates were injected
   intraperitoneally with STZ (40 mg · kg^−1 body weight in citrate
   buffer, pH 4.5, Sigma–Aldrich, St. Louis, MO, USA, #18883‐66‐4) for 5
   days to induce hyperglycemia. The mice were then given 10% sucrose for
   24 h to prevent hypoglycemic shock. One week after the injection, mice
   with fasting plasma glucose (FPG) ≥11.1 mmol L^−1 were selected for the
   following experiments. After continuous HFD‐feeding for 16 weeks, FPG,
   serum creatinine (Scr), blood urea nitrogen (BUN), kidney weight/body
   weight ratios (KW/BW), and urine albumin to creatinine ratios (urinary
   ACRs) were detected. When the above indicators far exceeded the normal
   values, the DKD model was successfully established.

Protocols In Vivo

   For the experiment of ASP (QYAOBIO, Hubei, CHN) intervention in DKD
   mice, twenty‐four 8‐week‐old C57BL/6 male mice were randomly allocated
   to four groups (six per group): normal diet (ND), ASP, DKD and DKD+ASP
   groups. After the model of T2DM was established by STZ/HFD, the mice in
   the ASP and DKD+ASP groups were intraperitoneally injected with ASP
   (0.5 µg · kg^−1· d^−1, ip) for 16 weeks. The mice in the ND and DKD
   groups were injected with saline daily. Meanwhile, the mice in the ND
   and ASP groups were fed with ND and those in the DKD and DKD+ASP groups
   were fed with HFD (60/100 kcal fat (soybean oil, lard), 20/100 kcal
   carbohydrate (500 kcal maltodextrin, 275 kcal sucrose) and 20/100 kcal
   protein, (Xietong, Inc., Nanjing, CHN) during the whole experiment,
   respectively.

   For the experiment of the effects of ASP^−/− on DKD mice, twelve
   8‐week‐old C57BL/6 male ASP^fl/fl mice were randomly divided into ND
   and DKD groups (six mice per group). In addition, twelve 8‐week‐old
   C57BL/6 male ASP^−/− C57BL/6 mice were randomly divided into ND and DKD
   groups (six mice per group). After the model of T2DM was established by
   STZ/HFD, the mice in ND (ASP^fl/fl+ND and ASP^−/−+ND) groups and DKD
   (ASP^fl/fl+DKD and ASP^−/−+DKD) groups were fed with ND and HFD for 16
   weeks, respectively.

   For the experiment of the effects of AASP (a neutralization antibody to
   ASP, for more information about AASP, please refer to our previously
   published paper^[ [204]^25 ^]), eighteen 8‐week‐old C57BL/6 male mice
   were randomly allocated to three groups (six mice per group), namely,
   ND, DKD, and DKD+AASP groups. After the model of T2DM was established
   by STZ/HFD, the mice in the DKD+AASP group were injected with AASP
   (0.5 µg · kg^−1· d^−1, ip), and those in the ND and DKD groups were
   injected with IgG (a control of AASP, ip) daily. The mice in the ND
   group were fed with ND, and the mice in the DKD (DKD, DKD+AASP) groups
   were fed with HFD during the whole experiment, respectively. Blood and
   urine samples of all 3 batches of mice were respectively collected
   every two weeks for the detection of the relevant indicators. The mice
   were sacrificed at the end of the experiments by inhaling 3% (v/v)
   isoflurane, and kidney and liver tissues were collected for further
   examinations.

Cell Culture

   Human renal tubular epithelial cell line (HK‐2 cells, ATCC, USA) was
   propagated in Dulbecco's Modified Eagle Medium/Nutrient Mixture F‐12
   (DMEM/F‐12) medium (Gibco Life Technologies, Carlsbad, CA, USA),
   supplemented with 10% fetal bovine serum (FBS, Tianhang, Zhejiang, CHN,
   #13011‐8611) and 1% penicillin/streptomycin (Gibco Life Technologies,
   Carlsbad, CA, USA). WT‐3T3‐L1 preadipocytes (ATCC, USA) and
   ASP‐deficient 3T3‐L1 preadipocytes (ASP^−/−, the construction method of
   ASP‐deficient cells and the methods of inducing 3T3‐L1 maturation can
   be referred to our previous paper^[ [205]^24 ^]) were conventionally
   cultured in H‐DMEM (Solarbio, Beijing, CHN) supplemented with 10% FBS
   (Tianhang, Zhejiang, CHN, #13011‐8611) and 1% penicillin/streptomycin
   (Gibco Life Technologies, Carlsbad, CA, USA). All cells were cultured
   in a humidified atmosphere containing 5% CO[2] at 37 °C. The cells were
   undergone distinct treatments, as specified in the Figure Legends.

Statistical analysis

   Data were presented as mean±SEM. Unpaired Student's t test (2‐sided)
   was used for statistical analysis between two groups. Nonparametric
   Mann‐Whitney test was applied for statistical analysis of tubular
   injury scores. One‐way or two‐way ANOVA followed by Tukey's multiple
   comparison test was used for multiple group comparisons. Pearson
   correlation analysis was used to calculate the correlation coefficient.
   P < 0.05 was considered statistically significant. GraphPad Prism 8.0
   (GraphPad Software) was used for statistical analysis. Detailed methods
   were provided in the Figure Legends and [206]Supporting Information
   section.

Conflict of Interest

   The authors declare no conflict of interest.

Author Contributions

   Q.Q.H., Q.R.H., and G.Z. conceived and designed the study. Q.Q.H. and
   X.X. assisted in the experimental designs. Q.Q.H., X.X., S.C., and Y.W.
   conducted the experiments and acquired the data. L.W., W.L., and C.L.
   analyzed the data. Q.Q.H. and Q.R.H. drafted and revised the
   manuscript. All authors approved the final version of the manuscript.

Supporting information

   Supporting Information
   [207]ADVS-12-e03259-s001.pdf^ (2.8MB, pdf)

Acknowledgements